Human Fas antigen is a polypeptide which is distributed in the surface of various cells and considered to be related to a type of the death of cells, called apoptosis. Homeostasis of the living body is skillfully regulated by the growth and differentiation of cells and the death thereof, and apoptosis is a mode of cell death which is discriminated from necrosis among modes of dying cells. It is known that the death of cells required for the maintenance of homeostasis of the living body, namely a case of cell death in which cells unnecessary for the living body are removed or virus-infected cells or tumor cells are attacked and removed by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells, is mainly based on apoptosis.
It has been remained unexplained for a long time about the true character of Fas antigen which is a monoclonal antibody obtained by immunizing mice with human fibroblasts, originally found by Yonehara, S. et al. (J. Exp. Med., vol. 169, pp. 1747-1756, 1989) as a cell surface antigen which is recognized by an anti-Fas antibody capable of inducing apoptosis in certain cells (Yonehara, S. et al.) and transfers a signal of apoptosis to the cells.
Recently, a Fas antigen gene has been cloned which revealed that the Fas antigen is a type I transmembrane glycoprotein which, according to its amino acid sequence, belongs to the NGFR (nerve growth factor receptor)/TNFR (TNF receptor) family that constitutes physiologically important cell surface membrane proteins (Itoh, N. et al., Cell, vol. 66, pp. 233-243, 1991). A mouse Fas antigen gene has also been cloned (Watanabe-Fukunaga, R. et al., J. Immunol., vol. 148, pp. 1274-1279, 1992), which confirmed that Fas antigen mRNA is expressed in the thymus, liver, lung, heart and ovary of mouse. Its expression in human has also been reported in various tissues and cells such as lymphocytes, hepatocytes, small intestine epithelial cells, skin keratinocytes and osteoblasts (Leithauser, F. et al., Lab. Invest., vol. 69, pp. 415-429, 1993)
The Fas antigen which was originally assumed to be present on the surface of cells has been detected in blood and culture supernatants, too, and its presence in the form of a soluble type Fas antigen (sFas) has also been found, so that their physiological functions and the like have become of interest in recent years.
Cheng, J. et al. (Science, vol. 263, pp. 1759-1762, 1994) have reported that they have found a mRNA molecule coding for a ΔTM type soluble form of Fas antigen, which seemed to encode a secretor molecule by the deletion of exon IV (corresponds to 14 base pairs of the extracellular region and 49 base pairs of the transmembrane region) due to alternative splicing, that concentration of the soluble type Fas antigen was high in sera of systemic lupus erythematosus (SLE) patients and that increase in the autologous mixed lymphocyte reaction was observed, together with increase in the number of splenocytes, when a chimera molecule composed of the extracellular region of mouse Fas antigen and the Fc moiety of human IgG was administered to mice. Also, Cascino, I. et al. (J. Immunol., vol. 154, pp. 2706-2713, 1995) have reported that at least three Fas antigen-encoding mRNA molecules exist, including the just described Fas antigen mRNA. Though their physiological functions, expression regions and the like are still unclear, it has been shown that the recombinant ΔTM type Fas antigen inhibits the cytotoxic activity of Fas ligand in a concentration-dependent manner and that most of the soluble type Fas antigens existing in sera of healthy people and SLE patients are ΔTM type Fas antigens encoded by splicing variants (Kayagaki, N. et al., Igaku-no Ayumi (Advance in Medical Science), vol. 174, pp. 1136-1140, 1995).
In addition, Kimura, K. et al. have recently cloned a rat Fas antigen gene and reported that two types of Fas antigen mRNA can be detected in rat liver mRNA based on the alternative splicing and that a mRNA molecule which encodes a peptide having smaller molecular weight is present in addition to the mRNA of membrane binding type Fas antigen (Biochemical and Biophysical Research Communications, vol. 198 (2), pp. 666-674 (1994)) and also have isolated and identified mutants (variants) of corresponding human Fas antigen (Japanese Patent Application Kokai No. 7-289266).
On the other hand, human Fas ligand is a polypeptide that has been reported by Nagata et al. to be a biological molecule that induces apoptosis of Fas antigen-expressing cells (Takahashi, T. et al., International Immunology, vol. 6, pp. 1567-1574, 1994). Human Fas ligand is a Type II membrane protein of TNF family with a molecular weight of about 40 kD. As in the case of TNF, human Fas ligand in the human body is estimated to be in the form of a trimer (Tanaka, M. et al., EMBO Journal, vol. 14, pp. 1129-1135, 1995). The extracellular domain of the human Fas ligand is highly homologous with the extracellular domain of rat Fas ligand (Suda, T. et al., Cell, vol. 75, pp. 1169-1178, 1993) and mouse Fas ligand (Takahashi, T. et al., Cell, vol. 76, pp. 969-976, 1994). The human Fas ligand recognizes not only human Fas antigen but also mouse Fas antigen, and induces the apoptosis. On the other hand, the rat Fas ligand and the mouse Fas ligand recognize the human Fas antigen to induce the apoptosis.
Apoptosis has called attention for its deep involvement in homeostasis of an organism. The homology of the Fas ligands among different species as mentioned above suggests the important role of the apoptosis mediated by the Fas ligand and the Fas antigen in the homeostasis of organism.
Recently, an interesting relation of abnormality of Fas ligand and Fas antigen with an autoimmune disease has been reported. In this report, there is suggested that MRL-1pr/1pr (a strain of model mouse for an autoimmune digease) has mutation in its Fas antigen gene, and apoptosis is not induced in the cells expressing such mutant Fas antigen gene (Watanabe-Fukunaga, R. et al., Nature, vol. 356, pp. 314-317, 1993; Adachi, M. et al., Proc. Natl. Acad. Sci. USA, vol. 90, 1993). In the meanwhile, there has also been reported that C3H-gld/gld (another strain of model mouse for an autoimmune disease) has mutation in its Fas ligand gene, and that the Fas ligand of the gld mouse has no apoptosis-inducing activity. The mutation in the Fas ligand gene of the gld mouse is a point mutation, and as a result of such point mutation, 7th amino acid from the C terminal of the extracellular domain of the Fas ligand is replaced with another amid acid (Tomohiro Takahashi, et al., Cell, vol. 76, pp. 969-976, 1994). The Fas ligand of the gld mouse as described above is incapable of binding with the Fas antigen (Fred Ramsdell, et al., Eur. J. Immunol., vol. 24, pp. 928-933, 1994).
Also, in the case of SLE patients, increase in the level of a soluble type Fas antigen in blood (Cheng J. et al., Science, vol. 263, pp. 1759-1762, 1994) and increase in the expression of a Fas antigen on the surface of lymphocytes (Amasaki T. et al., Clin. Exp. Immunol., vol. 99, pp. 245-250, 1995) have been reported, and it has been suggested about a possibility that abnormality of a Fas antigen gene accompanied by the abnormal expression and function of the Fas antigen is concerned also in lymphocytosis syndrome (Rieux Laucat et al., Science, vol. 268, pp. 1347-1349, 1995, and Fisher G. H. et al., Cell, vol. 81, pp. 935-946, 1995).
The finding as described above resulted in the hypothesis that some autoimmune diseases are induced by the abnormality of the Fas antigen or the Fas ligand, namely, by the autoreactive T cells remaining in the body that should have been removed from the body by undergoing apoptosis if the cells had been normal.
Recently, it is conceived that abnormal propagation and articular cytoclasis of synovial membrane that takes place in articular rheumatism is also induced by the failure of normal apoptosis of the cell. Kobayashi, N. et al. further estimates that reduction in the number of T cells upon infection by AIDS virus is mediated by the Fas ligand since expression of the Fas antigen on the T cell membrane is induced upon infection by the AIDS virus (Nikkei Science, vol. 6, pp. 34-41, 1993).
Participation of apoptosis by Fas ligand has been suggested also in viral fulminant hepatitis and the like diseases (Nagata, S. et al., Immunol. Today, vol. 16, pp. 39-43, 1995). Thus, as the relationship between Fas antigen-mediated apoptosis and diseases has been revealed, great concern has been directed toward the application of Fas ligand or Fas antigen to the treatment of diseases which are accompanied by abnormal apoptosis, namely the aforementioned autoimmune diseases, rheumatism, AIDS and the like.
Examples of the substance which inhibits Fas antigen-mediated apoptosis so far reported include a ΔTM type Fas antigen, a fusion protein of the extracellular region of Fas antigen with the Fc region of immunoglobulin G (IgG) and a fragment of anti-Fas antibody (Dhein J. et al., Nature, vol. 373, pp. 438-441, 1995) and an anti-Fas antagonist antibody (Alderson M. A. et al., Int. Immunol., vol. 6, pp. 1799-1806, 1994).
Nagata et al. have also reported that they succeeded in obtaining an antibody against the Fas ligand, and that such antibody was capable of suppressing the apoptosis (Masato Tanaka, et al., EMBO Journal, vol. 14, pp. 1129-1135, 1995; International Patent Application Laid-Open No. WO95/13293).
As described in the foregoing, as Fas antigens have been isolated, it has been revealed that apoptosis is induced via these Fas antigens in the living body and the Fas antigen-mediated apoptosis is concerned in various diseases, so that it is considered that a substance capable of controlling the Fas antigen-mediated apoptosis will be useful in the prevention, treatment and diagnosis of diseases in which apoptosis is presumably concerned. Though usefulness of a pharmaceutical preparation in the living body should be evaluated by synthetically taking its activity, biological behavior, safety, side effects and the like into consideration, a substance having particularly high activity will exert its efficacy at a low dosage in the living body so that it will have high industrial availability. Because of this, great concern has been directed toward the development of an improved novel Fas antigen derivative, especially a Fas antigen derivative having more high activity, and the elucidation of its characteristics, particularly, when its application to the living body such as human treatment is taken into consideration, a novel Fas antigen derivative capable of controlling the Fas antigen-mediated apoptosis at more smaller dosage is expected from the viewpoint of efficacy and safety.
In addition, acquisition of a novel Fas antigen derivative is also important for the elucidation of, for example, interaction of Fas antigen and Fas ligand and mechanism of the Fas antigen-mediated apoptosis.
It is known that a polypeptide can show its activity even in the form of its partial fragment when the fragment preserve its intrinsic structure at the active site. On the contrary, however, many cases are also known in that the activity of a polypeptide is changed, or lost in an extreme case, by a mutation or deletion of even one amino acid in the polypeptide.
Thus, an object of the present invention is to provide the field of medicaments with a novel Fas antigen derivative whose activity and the like are improved and with a novel DNA fragment which encodes the same. The present invention also provides a recombinant DNA molecule and a transformant, which contains said DNA fragment, and a method for the production of said novel Fas antigen derivative.